To successfully eliminate malaria, which continues to be responsible for 8% of all the deaths of children under five, new malaria control strategies are needed, including transmission blocking (TxB) vaccines. Toward this goal, we have recently produced correctly-folded P. falciparum gametocyte surface protein P48/45 (Pfs48/45) in a form that is ready for GMP production (GMZ3) and induces antibodies that disrupt parasite infectivity to mosquitoes in a standard membrane feed using in vitro culture-adapted parasites. We hypothesize that anti-GMZ3 antibodies will also block the ability of natural parasite isolates to infect mosquitoes and that naturally acquired TxB immunity will correlate with the presence of antibodies that recognize GMZ3 indicating that it is a good vaccine candidate. To test this we propose to AIM 1) evaluate the ability of anti-GMZ3 antibodies produced in rats to block the infectivity of natural isolates to mosquitoes. We will also evaluate AIMS 2 & 3) whether natural parasite exposure induces Pfs48/45/GMZ3 antibodies that contribute to transmission-blocking immunity. Pfs48/45 is one of four Plasmodium proteins (Pfs48/45 and P230, P25 and P28) that are the targets of monoclonal antibodies that effectively block malaria Tx and have been targeted as vaccine candidates. All these proteins are relatively cysteine-rich with multiple disulfide bonds resulting in antibody epitopes that are dependent on tertiary structure rather than linear amino acid sequence. Only P25 has advanced to a Phase Ia clinical trial, but further development has been slow due to limited immunogenicity and the lack of boosting during a natural malaria infection. Both Pfs48/45 and Pfs230 are expressed as the parasite undergoes sexual differentiation into gametocytes in the human host and naturally occurring anti-Pfs48/45 antibodies have been found after exposure to malaria. This suggests that the immune response against a Pfs48/45 or Pfs230-based TxB vaccine could be boosted during a natural infection enhancing the efficacy of the vaccine. The project proposed here will directly evaluate the potential of GMZ3 as a vaccine candidate against natural isolates. It augments ongoing epidemiologic and clinical safety studies by directly comparing transmission-blocking activity with gametocyte prevalence, genetic variation and anti-rPfs48/45.10C antibody titers and will provide critical information to direct the further development of an effective transmission-blocking vaccine.